1. Field of the Invention
This invention lies in the field of hemocytometry and systems in general for the counting of cells in biological tissue or fluids.
2. Description of the Prior Art
The ability to distinguish between live cells and dead cells in biological tissue or fluid offers information of value in both diagnostics and research. The distinction is of value in assessing microbial infestations, for example, since only the live cells in a microbial population possess metabolic and reproductive activity, and when the cells are pathogenic microorganisms, only the live cells present a potential health risk. The distinction is also of value in investigations of the efficacy or mechanism of action of drugs and pharmaceuticals, since determinations of cell viability or death are often a part of such investigations. Live cells must also be distinguished from dead cells in certain lines of research that involve investigations of morphological characteristics of cells, since the results of these investigations can be obscured when the characteristics of interest are masked by phenotypic characteristics of dead cells.
One means of distinguishing between live and dead cells is by assaying for DNA, since DNA is commonly associated only with live cells. This method is not reliable, however, since DNA can persist in a cell for several weeks after cell death. Other methods include capturing images of cells that have been treated with a contrast agent that associates with a marker that is present in only live or only dead cells. The treatment is an extra step, however, and is not one that automated instruments that are currently in existence can be readily adapted to incorporate. Still further methods include the use of vital stains that cause dead cells to absorb incident light and that thereby allow one to eliminate dead cells from the total cell count. When dead cells are treated with vital stains, however, the outlines of the cells are often indiscernible and multiple cells that overlap or abut each other can be miscounted as single cells, while single cells of irregular shapes can be mistaken as two or more cells. Live cells are relatively easy to differentiate from each other, even when they are in clumps or of dissimilar sizes, because live cells have bright cores and dark contours, resulting in contrast between the cores and the cell outlines that permits individual cell recognition by computer software or the human brain. No such contrast is present in dead cells since both the cell interiors and the cell contours are dark. The declumping of dead cells for counting purposes is therefore nearly impossible. Certain cell counters presently available from commercial suppliers have a large field of view in combination with limited resolution. Together, these features make it difficult to differentiate live cells from dead cells, and the difficulty is aggravated when the dead cells are clumped together.